Skeletal muscle insulin resistance is an underlying pre-requisite to the development of diabetes, however the fundamental mechanisms that underlie insulin resistance are multi-factorial and poorly understood. Increased skeletal muscle diacylglycerol (DAG) concentration is one mechanism influencing insulin sensitivity in humans. All studies published in humans to date have considered muscle DAG as a homogeneous group of molecules with equal ability to impact insulin action. However, there are over 20 abundant species of DAG based on the most prevalent fatty acids binding to the first two carbons of the molecule. In vitro data suggest fatty acids on DAG may impact the ability to increase protein kinase C (PKC) activity, which is the currently accepted mechanism by which DAG impacts insulin sensitivity. However, little is known regarding the molecular composition or cellular location of DAG in human muscle. Further, it is not known if different molecular species or cellular localization of DAG influence PKC activity, and therefore, insulin sensitivity. The hypothesis we are testing in this study is that the composition and localization of DAG impacts insulin sensitivity in humans. Our preliminary data suggest individuals who are insulin resistant have increased amounts of DAG containing saturated fatty acids compared to insulin sensitive individuals. Additionally, our data suggest endurance training decreases the saturated fat composition of DAG. The aim of this proposal is to determine: 1) if saturation of DAG is related to insulin sensitivity in humans, and 2) if the cellular localization of molecular species of DAG are related to PKC activation and insulin sensitivity in humans, and 3) to determine if alterations in DAG composition without a change in DAG concentration influences PKC activation and insulin sensitivity in cell culture. These studies are hoped to advance our understanding of the interplay between muscle DAG and insulin resistance, allowing development of better therapeutic strategies to prevent and treat diabetes. PUBLIC HEALTH RELEVANCE: Narrative The prognosis for individuals with type 2 diabetes remains poor. It is vital to understand the pathophysiology of diabetes to develop better therapeutic targets for treatment and prevention. These studies may allow a better understanding of factors which promote and prevent insulin resistance in humans so that better therapeutic targets can be developed.